Unraveling the Acidithiobacillus caldus complete genome and its central metabolisms for carbon assimilation

https://doi.org/10.1016/j.jgg.2011.04.006Get rights and content

Abstract

Acidithiobacillus caldus is one of the dominant sulfur-oxidizing bacteria in bioleaching reactors. It plays the essential role in maintaining the high acidity and oxidation of reduced inorganic sulfur compounds during bioleaching process. In this report, the complete genome sequence of A. caldus SM-1 is presented. The genome is composed of one chromosome (2,932,225 bp) and four plasmids (pLAtc1, pLAtc2, pLAtc3, pLAtcm) and it is rich in repetitive sequences (accounting for 11% of the total genome), which are often associated with transposable genetic elements. In particular, twelve copies of ISAtfe and thirty-seven copies of ISAtc1 have been identified, suggesting that they are active transposons in the genome. A. caldus SM-1 encodes all enzymes for the central metabolism and the assimilation of carbon compounds, among which 29 proteins/enzymes were identifiable with proteomic tools. The SM-1 fixes CO2 via the classical Calvin–Bassham–Benson (CBB) cycle, and can operate complete Embden-Meyerhof pathway (EMP), pentose phosphate pathway (PPP), and gluconeogenesis. It has an incomplete tricarboxylic acid cycle (TCA). Four putative transporters involved in carbohydrate uptake were identified. Taken together, the results suggested that SM-1 was able to assimilate carbohydrates and this was subsequently confirmed experimentally because addition of 1% glucose or sucrose in basic salt medium significantly increased the growth of SM-1. It was concluded that the complete genome of SM-1 provided fundamental data for further investigation of its physiology and genetics, in addition to the carbon metabolism revealed in this study.

Introduction

Acidithiobacillus caldus, a moderately thermoacidophilic and obligately chemolithotrophic r-Proteobacterium with an optimal growth temperature of 40–45°C and pH of 2–2.5 (Kelly and Wood, 2002), was reported to be the dominant sulfur-oxidizing bacterium in biomining (Xia et al., 2009, Zhou et al., 2009, Zeng et al., 2010, Spolaore et al., 2011) and it was also found to be associated with acid mine drainage (Kamimura et al., 2010). The main roles of A. caldus in biomining processes include: 1) to oxidize elemental sulfur and reduced inorganic sulfur compounds (RISCs), thus produces the acidity that is essential for biomining and 2) to remove the accumulated elemental sulfur that would otherwise retard the oxidation of ores (Dopson and Lindström, 1999, Watling, 2006). RISC oxidation has been the focus of A. caldus investigations (Mangold et al., 2011). It was shown that RISC oxidation in A. caldus is coupled to ATP generation via electron transport phosphorylation (Dopson et al., 2002). A periplasmic tetrathionate hydrolase (TetH) was purified and characterized from A. caldus (Bugaytsova and Lindström, 2004). The TetH gene was cloned and co-transcribed with a thiosulfate quinone oxidoreductase gene (doxD) from a common genetic cluster in A. caldus (Rzhepishevska et al., 2007). Further, a gene encoding a sulfur oxygenase reductase that oxidizes elemental sulfur was identified in A. caldus (Chen et al., 2007).

Comparing to the knowledge of RISC oxidation and energy metabolism, much less was done to study CO2 fixation and central metabolism of carbon compounds in A. caldus in the past years. Uptake and fixation of CO2 at an extremely acidic condition has been much overlooked until recently a form II Rubisco was presented in Acidithiobacillus ferrooxidans that could promote the ability to fix CO2 at different concentrations of CO2 (Esparza et al., 2010). Considering the extremely low pH (thus CO2 would be existing mainly as dissolved gas instead of bicarbonates), as well as the oligotrophic nature of the environments in which Acidithiobacillus species lives, it was expected that Acidithiobacillus species might possess novel strategies to sequestrate carbon compounds from environments for growth.

In this report, we present the complete genome sequence of A. caldus SM-1, which was isolated from a pilot bioleaching reactor (Liu et al., 2010). Genome analysis was focused on the central metabolisms for carbon compounds and the results were validated by applying proteomic tools. We concluded that SM-1 was able to uptake and assimilate a variety of organic compounds for growth.

Section snippets

Preparation of genomic DNA and DNA sequencing

A. caldus SM-1 was cultivated at 45°C in 9 K medium (Silverman and Lundgren, 1959) supplemented with 2% elemental sulfur and 0.05% yeast extract. Cells were harvested in the stable-phase and sulfur was removed from the cells by differential centrifugation. Genomic DNA was extracted by phenol–chloroform methods as described previously (Marmur, 1961).

The A. caldus SM-1 genome was sequenced by using the Roche 454 Genome Sequencer FLX instrument (454 Life Science, Branford, USA). A total of 522,895

General features of the A. caldus SM-1 genome

The complete SM-1 genome was composed of one chromosome and four plasmids (pLAtc1, pLAtc1, pLAtc3, and pLAtcm), giving a total genome size of 3,237,599 bp (Fig. 1 and Table 1). The circular chromosome comprised 91% of the genome with an average GC content of 61%. There were 2880 putative ORFs, of which 1938 ORFs could be functionally annotated, 744 were conserved hypothetical proteins while 198 were unique hypothetical proteins. Six hundred and five (21%) ORFs were predicted as secretory

Discussion

The occurrence of large numbers of transposons can result in genome instability, as reported for the Acetobacter pasteurianus genome (Azuma et al., 2009). The SM-1 strain encodes 198 transposable genetic elements including 37 copies of ISAtc1 and 12 copies of ISAtfe. Based on our analysis, these transposable genetic elements have exerted significant effects on the SM-1 genome stability including gene inactivation, gene loss and gene acquisition: seven genes were interrupted by insertion of a

Acknowledgements

The work was supported by the National Science Foundation of China (No. 30870039) and the National Basic Research Program of China (973 Program, No. 2010CB630903). Constructive suggestion during preparation of the manuscript from Prof. Y. Tao at Institute of Microbiology, Chinese Academy of Sciences is highly acknowledged.

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